Effect of time of artificial insemination and supplemental estradiol on reproduction of lactating dairy cows

Our objectives were to compare reproductive responses of dairy cows receiving timed artificial insemination (AI) either at 48 or 72 h after induction of luteolysis and supplemented or not with estradiol cypionate (ECP). Holstein cows (971) had their estrous cycles presynchronized with injections of PGF_2α at 37 and 51 d in milk (DIM) and then received an injection of GnRH at 64 DIM and an injection of PGF_2α at 71 DIM. Cows were then assigned to a 2 × 2 factorial randomized block experiment; cows in the CoSynch 48 h (CoS48) received a final injection of GnRH concurrent with timed AI 48 h after PGF_2α, whereas cows in the CoSynch 72 h (CoS72) received GnRH and timed AI 72 h after PGF_2α. Half of the cows in each CoSynch protocol received an injection of 1 mg of ECP 24 h after PGF_2α. Therefore, the 4 treatments were as follows: CoS48-NECP (n = 240), CoS72-NECP (n = 246), CoS48-ECP (n = 245), and CoS72-ECP (n = 240). Blood was sampled at 7 d before and at the first GnRH of the CoSynch from all cows for analysis of progesterone concentration in plasma. Cows were classified as anovular when progesterone was less than 1.0 ng/mL in both samples. Blood was also sampled during proestrus from a subset of 123 cows to measure concentrations of estradiol and at 7 d after timed AI to measure concentrations of progesterone. Ovaries from the same subset of 123 cows were examined by ultrasonography to determine ovulatory follicle diameter and incidence of ovulation. Pregnancy was diagnosed at 40 and 68 d after AI. Prevalence of cyclic cows was 72.4% and was similar among treatments. Concentrations of estradiol increased after ECP treatment and at 72 h of proestrus with CoS72. Pregnancy at 40 and 68 d after AI and pregnancy loss were not affected by timing of AI or supplemental ECP. Delaying timed AI to 72 h and supplementation with ECP increased the proportion of cows displaying estrus at AI, and cows detected in estrus had increased pregnancy per AI associated with improved ovulation and increased postovulatory progesterone concentration. These results indicate that extending the proestrus by delaying timed AI from 48 to 72 h plus supplemental ECP, despite increased expression of estrus at timed AI, did not improve reproductive performance of lactating dairy cows at first AI.     

Effect of interval between induction of ovulation and artificial insemination (AI) and supplemental progesterone for resynchronization on fertility of dairy cows subjected to a 5-d timed AI program

Objectives were to investigate 2 intervals from induction of ovulation to artificial insemination (AI) and the effect of supplemental progesterone for resynchronization on fertility of lactating dairy cows subjected to a 5-d timed AI program. In experiment 1, 1,227 Holstein cows had their estrous cycles presynchronized with 2 injections of PGF_2α at 46 and 60 d in milk (DIM). The timed AI protocols were initiated with GnRH at 72 DIM, followed by 2 injections of PGF_2α at 77 and 78 DIM and a second injection of GnRH at either 56 (OVS56) or 72 h (COS72) after the first PGF_2α of the timed AI protocols. All cows were time-inseminated at 72 h after the first PGF_2α injection. Pregnancy was diagnosed on d 32 and 60 after AI. In experiment 2, 675 nonpregnant Holstein cows had their estrous cycles resynchronized starting at 34 d after the first AI. Cows received the OVS56 with (RCIDR) or without (RCON) supplemental progesterone, as an intravaginal insert, from the first GnRH to the first PGF_2α. Pregnancy diagnoses were performed on d 32 and 60 after AI. During experiment 2, subsets of cows had their ovaries scanned by ultrasonography at the first GnRH, the first PGF_2α, and second GnRH injections of the protocol. Blood was sampled on the day of AI and 7 d later, and concentrations of progesterone were determined in plasma. Cows were considered to have a synchronized ovulation if they had progesterone <1 and >2.26 ng/mL on the day of AI and 7 d later, respectively, and if no ovulation was detected between the first PGF_2α and second GnRH injections during resynchronization. In experiment 1, the proportion of cows detected in estrus at AI was greater for COS72 than OVS56 (40.6 vs. 32.4%). Pregnancy per AI (P/AI) did not differ between OVS56 (46.4%) and COS72 (45.5%). In experiment 2, cows supplemented with progesterone had greater P/AI compared with unsupplemented cows (51.3 vs. 43.1%). Premature ovulation tended to be greater for RCON than RCIDR cows (7.5 vs. 3.6%), although synchronization of the estrous cycle after timed AI was similar between treatments. Timing of induction of ovulation with GnRH relative to insemination did not affect P/AI of dairy cows enrolled in a 5-d timed AI program. Furthermore, during resynchronization starting on d 34 after the first AI, supplementation with progesterone improved P/AI in cows subjected to the 5-d timed AI protocol.     

Altering the time of the second gonadotropin-releasing hormone injection and artificial insemination (AI) during Ovsynch affects pregnancies per AI in lactating dairy cows

Based on previous research, we hypothesized that Cosynch at 72 h [GnRH−7 d−PGF_2α;−72 h−GnRH + artificial insemination (AI)] would result in a greater number of pregnancies per AI (P/AI) than Cosynch at 48 h. Further, we hypothesized that P/AI would be improved to a greater extent when GnRH was administered at 56 h after PGF_2α; before AI at 72 h due to a more optimal interval between the LH surge and AI. Nine hundred twenty-seven lactating dairy cows (n = 1,507 AI) were blocked by pen, and pens rotated through treatments. All cows received GnRH followed 7 d later by PGF_2α; and then received one of the following: 1) GnRH + timed AI 48 h after PGF_2α; (Cosynch-48); 2) GnRH 56 h after PGF_2α; + timed AI 72 h after PGF_2α; (Ovsynch-56); or 3) GnRH + timed AI 72 h after PGF_2α; (Cosynch-72). Pregnancy diagnoses were performed by ultrasound at 31 to 33 d post-AI and again at 52 to 54 d post-AI. Overall P/AI were similar for the Cosynch-48 (29.2%) and Cosynch-72 (25.4%) groups. The Ovsynch-56 group had a greater P/AI (38.6%) than Cosynch-48 or Cosynch-72. Presynchronized first-service animals had greater P/AI than cows at later services in Cosynch-48 (36.2 vs. 23.0%) and Ovsynch-56 (44.8 vs. 32.7%) but not in Cosynch-72 (24.6 vs. 26.2%). Similarly, primiparous cows had greater P/AI than multiparous cows in Cosynch-48 (34.1 vs. 22.9%) and Ovsynch-56 (41.3 vs. 32.6%), but not Cosynch-72 (29.8 vs. 25.3%). In conclusion, we found no advantage to Cosynch at 72 h vs. 48 h. In contrast, we found a clear advantage to treating with GnRH at 56 h, 16 h before a 72 h AI, probably because of more-optimal timing of AI before ovulation.     

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AI (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AI on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF_2α and removal of the CIDR on d 5, and GnRH concurrent with timed AI on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF_2α injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF_2α did not differ between treatments and averaged 87.1%. Progesterone on the day of AI was greater for GnRH than control (0.50 ± 0.07 vs. 0.28 ± 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GnRH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF_2α and removal of the CIDR on d 5, and either GnRH 56 h after PGF_2α and AI 16 h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF_2α (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but CO S72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF_2α injection 5 d later. Moreover, extending the proestrus by delaying the final GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.     

Reproductive performance of grazing dairy cows following presynchronization and resynchronization protocols

Objectives were to compare the effect of presynchronization and resynchronization methods on fertility responses of grazing dairy cows at first and second artificial insemination (AI) and pregnancy rate during the entire breeding season. Lactating dairy cows (n = 1,263) in 2 seasonal grazing farms were blocked, within farm, by parity, breed and days in milk. Within each block, cows were randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial with 2 presynchronization and 2 resynchronization treatments. Cows had their estrous cycles presynchronized with either a PGF_2α-based program (Presynch) consisting of 2 injections of PGF_2α administered 14 d apart and starting the timed AI protocol 11 d later, or with a PGF_2α-GnRH-based presynchronization program (G6G) consisting of an injection of PGF_2α, followed 3 d later by an injection of GnRH and starting the timed AI protocol 6 d later. All cows received the first insemination on the same day, which was considered study d 0 and also d 0 of the breeding season. All cows received the 5-d timed AI protocol that consisted of GnRH on d −8, PGF_2α on d −3 and −2, and GnRH + timed AI on d 0. Blood was sampled and analyzed for progesterone on d −8. On d 12, cows in each presynchronization treatment either remained as untreated controls (RCON) or received a controlled internal drug-release (CIDR) insert containing progesterone for 7 d (RCIDR). Estrus was observed daily starting on d 19 and cows in estrus were inseminated on the same day. On d 35, bulls were placed with the cows for an additional 65 d, completing a 100-d breeding season. Holstein cows were less likely to have progesterone ≥1 ng/mL on d −8, and had less expression of estrus and pregnancy per AI (P/AI), which resulted in a slower rate of pregnancy and a smaller proportion of pregnancy at the end of the study than did Jersey or crossbred cows. In addition, body condition, days in milk, and plasma progesterone concentration at the first GnRH injection of the timed AI protocol had marked effects on the reproductive performance of lactating grazing dairy cows. A greater proportion of G6G cows had progesterone ≥1ng/mL at the first GnRH injection of the timed AI protocol compared with Presynch cows (82.0 vs. 74.3%). Presynchronization treatment did not influence P/AI, but cows in G6G had increased risk of pregnancy loss between d 30 and 65 after the first AI (12.9 vs. 8.1%). Nevertheless, an interaction between presynchronization and ovarian status was observed, and cows initiating the timed AI with progesterone ≥1 ng/mL had greater P/AI when previously treated with Presynch than G6G. On the other hand, G6G benefited P/AI of cows initiating the timed AI with progesterone <1 ng/mL. Resynchronization with RCIDR altered the pattern of return to estrus, but it did not increase the rate of re-insemination and decreased the proportion of pregnant cows at the end of the 100-d breeding period (80.6 vs. 84.4%).     

Dose frequency of prostaglandin F2α administration to dairy cows exposed to presynchronization and either 5- or 7-day Ovsynch program durations: Ovulatory and luteolytic risks

We hypothesized (1) that neither duration of the Ovsynch program nor dose frequency of PGF_2α would change the proportion of cows with complete luteolysis (progesterone <0.4 ng/mL 72 h after PGF_2α) and (2) that the additional GnRH treatment administered as part of a presynchronization program would not alter the proportion of anovulatory cows starting the timed artificial insemination (AI) program compared with an alternative shorter presynch program including only 1 GnRH treatment. Lactating Holstein cows (n = 406) were milked 3 times daily and enrolled in a 2 × 2 × 2 factorial experiment consisting of 8 treatments before the first postpartum AI. Treatments were used to test ovulatory, luteal, and luteolytic outcomes to 3 main effects: (1) 2 GnRH-PGF_2α presynchronization programs (PG-3-G vs. Double Ovsynch), (2) 2 Ovsynch program durations [5 d: GnRH (GnRH-1)–5 d–PGF_2α–24 h–PGF_2α–32 h–GnRH (GnRH-2)–16 h–timed AI; 7 d: GnRH-1–7 d–PGF_2α–56 h–GnRH-2–16 h–timed AI], and (3) 2 PGF_2α dose frequency treatments (2 × 25 mg) 24 h apart versus 1 dose (1 × 50 mg) of PGF_2α administered 72 h before timed AI. The presynchronization treatments of PG-3-G and Double Ovsynch had no effect on the proportion of cows with luteal function at the onset of the Ovsynch treatments (87.9 vs. 86.2%). Although ovulatory responses were similar after GnRH-1 (>60%), Double Ovsynch cows tended to have greater ovulatory responses than PG-3-G after GnRH-2 (95.3 vs. 90.6%). The 2 × 25-mg doses of PGF_2α and the 1 × 50-mg dose induced luteolysis in both Ovsynch treatment durations, but the 1 × 50-mg dose was less effective in the 5-d program. More pregnancy per AI (P/AI; 49.2%) tended to occur in the PG-3-G cows in the 7-d program compared with the other treatment combinations (range: 32.4–37.4%; Ovsynch × presynch interaction). In addition, an Ovsynch × PGF_2α dose frequency interaction resulted in cows receiving the 1 × 50-mg dose in the 7-d program having the greatest P/AI (46.1%) and cows receiving the 1 × 50-mg dose in the 5-d program having the least P/AI (30.6%). We conclude that complete luteolysis was less effective in the 5-d program when the 1 × 50-mg dose was applied, but both PGF_2α dose frequencies (1 × 50 mg and 2 × 25 mg 24 h apart) effectively induced complete luteolysis in the 7-d program. Treatments producing complete luteolysis tended to be related to subsequent pregnancy outcomes.     

Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows

Two experiments evaluated the influence of follicular wave at artificial insemination (AI) on fertility of dairy cows. In experiment 1, data from 5,607 lactating cows enrolled in estrous and ovulation synchronization programs for AI were evaluated. Cows’ blood was analyzed for progesterone 7 to 14 d apart, with the second sample collected on the day of the first GnRH (GnRH1) of the synchronization protocol. Cows were classified as cyclic if progesterone was ≥1 ng/mL in at least 1 of the 2 samples and as anovular if both samples were <1 ng/mL. Cyclic cows were categorized as low (CLOW; < 1 ng/mL) or high (CHIGH; ≥ 1 ng/mL) progesterone on the day of GnRH1, which would result in ovulation of the dominant follicle of the first (FW) and second (SW) follicular waves, respectively, at AI. Pregnancy per AI (P/AI) was determined 30 and 53 d after AI. In experiment 2, 220 cyclic Holstein cows received 2 injections of PGF_2α administered 14 d apart. The Ovsynch protocol (d 0 GnRH, d 7 PGF_2α, d 9 GnRH, d 9.5 timed AI) was initiated either 3 or 10 d after the second PGF_2α of the presynchronization to result in insemination to the FW or SW dominant follicles. Blood was analyzed for progesterone and ovaries were scanned to determine ovulatory responses and follicle diameter. Pregnancy was determined on d 32 and 67 after timed AI. In experiment 1, P/AI on d 30 was greater for CHIGH cows than for CLOW and anovular cows (43.0, 31.3, and 29.7%, respectively), but because of pregnancy loss, P/AI on d 53 was lowest for anovular cows. Proportions of cows with short reinsemination intervals differed among groups and were 7.1, 15.7, and 11.9% for CHIGH, CLOW, and anovular cows, respectively. Pregnancy loss was greater for anovular cows than for CLOW cows (15.0 vs. 10.0%) and was intermediate for CHIGH cows (13.5%). In experiment 2, 9.8 and 97.2% of the FW and SW cows, respectively, had progesterone ≥1 ng/mL at GnRH1. Concentrations of progesterone at the GnRH1 and PGF_2α injections of the Ovsynch protocol were greater for SW cows than FW cows. Pregnancy per AI was greater for SW cows than for FW cows (41.7 vs. 30.4%) despite less ovulation to GnRH1 in SW cows than in FW cows (78.7 vs. 88.4%). Collectively, these data indicate that follicular wave of the ovulatory follicle and not cyclic status caused the greatest reduction in P/AI in dairy cows. Whether the culprit is the follicle itself or the hormonal milieu characteristic of the first follicular wave and the early stage of the estrous cycle remains to be elucidated. Synchronization programs that induced ovulation of the FW follicle at AI reduced P/AI in lactating dairy cows, and ovulation of the FW follicle, or development of the ovulatory follicle under low progesterone concentrations, or both, might be mechanisms for reduced fertility in anovular cows.     

Timed artificial insemination programs during the summer in lactating dairy cows: Comparison of the 5-d Cosynch protocol with an estrogen/progesterone-based protocol

The objective of this study was to compare a GnRH-based to an estrogen/progesterone (E2/P4)-based protocol for estrous cycle synchronization and fixed timed artificial insemination (TAI), both designed for synchronization of ovulation and to reduce the period from follicular emergence until ovulation in cows with a synchronized follicular wave. A total of 1,190 lactating Holstein cows (primiparous: n = 685 and multiparous: n = 505) yielding 26.5 ± 0.30 kg of milk/d at 177 ± 5.02 d in milk were randomly assigned to receive one of the following programs: 5-d Cosynch protocol [d −8: controlled internal drug release (CIDR) + GnRH; d −3: CIDR removal + PGF_2α; d −2: PGF_2α; d 0: TAI + GnRH] or E2/P4 protocol (d −10: CIDR + estradiol benzoate; d −3: PGF_2α; d −2: CIDR removal + estradiol cypionate; d 0: TAI). Rectal temperature and circulating progesterone (P4) were measured on d −3, −2, 0 (TAI), and 7. The estrous cycle was considered to be synchronized when P4 was ≥1.0 ng/mL on d 7 in cows that had luteolysis (P4 ≤0.4 ng/mL on d 0). To evaluate the effects of heat stress, cows were classified by number of heat stress events: 0, 1, and 2-or-more measurements of elevated body temperature (≥39.1°C). Pregnancy success (pregnancy per artificial insemination, P/AI) was determined at d 32 and 60 after TAI. The cows in the 5-d Cosynch protocol had increased circulating P4 at the time of PGF_2α injection (2.66 ± 0.13 vs. 1.66 ± 0.13 ng/mL). The cows in the E2/P4 protocol were more likely to be detected in estrus (62.8 vs. 43.4%) compared with the cows in the 5-d Cosynch protocol, and expression of estrus improved P/AI in both treatments. The cows in the 5-d Cosynch protocol had greater percentage of synchronized estrous cycle (78.2%), compared with cows in the E2/P4 protocol (70.7%). On d 60, the E2/P4 protocol tended to improve P/AI (20.7 vs. 16.7%) and reduced pregnancy loss from 32 to 60 d (11.0 vs. 19.6%), compared with the 5-d Cosynch protocol. In cows withtheir estrous cycle synchronized, the E2/P4 protocol had greater P/AI (25.6 vs. 17.7%) on d 60 and lower pregnancy loss from 32 to 60 d (6.7 vs. 21.7%) compared with cows in the 5-d Cosynch protocol. Follicle diameter affected pregnancy loss from 32 to 60 d only in the cows in the 5-d Cosynch protocol, with smaller follicles resulting in greater pregnancy loss. Pregnancy per AI at d 60 was different between protocols in the cows with 2 or more measurements of heat stress (5-d Cosynch = 12.2% vs. E2/P4 = 22.8%), but not in the cows without or with 1 heat stress measurement. In conclusion, the 5-d Cosynch protocol apparently produced better estrous cycle synchronization than the E2/P4 protocol but did not improve P/AI. The potential explanation for these results is that increased E2 concentrations during the periovulatory period can improve pregnancy success and pregnancy maintenance, and this effect appears to be greatest in heat-stressed cows when circulating E2 may be reduced.     

The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch,compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF_2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF_2α–14 d–PGF_2α–10 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. Treated cows received PGF_2α and GnRH–7 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. All cows received a supplemental injection of PGF_2α 24 h after the PGF_2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF_2α of Ovsynch. The administration of PGF_2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF_2α of Ovsynch, total luteal area (mm²), or serum concentrations of P4 at time of PGF_2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF_2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.     

Progesterone, Follicular, and Estrual Responses to Progesterone-Based Estrus and Ovulation Synchronization Protocols at Five Stages of the Estrous Cycle

The objective was to monitor changes in ovarian status in heifers exposed to a progesterone insert with or without concurrent GnRH injection. Estrus was manipulated in 283 heifers (31 breeding clusters) by administering GnRH, progesterone, and PGF_2α at 5 stages of the estrous cycle. Estrus was presynchronized with a progesterone insert (CIDR) for 7 d before PGF_2α was administered 24 h before insert removal. Successive clusters of heifers were assigned to treatments (2 heifers per treatment) on cycle d 2, 5, 10, 15, and 18. Treatments consisted of a progesterone insert (d 0) for 7 d plus: 1) PGF_2α on d 6, 24 h before insert removal (early PGF); 2) GnRH on d 0 + early PGF_2α (GnRH + early PGF); 3) PGF_2α at insert removal (late PGF); and 4) GnRH on d 0 + late PGF (GnRH + late PGF). Controls received GnRH on d 0 and PGF_2α on d 7. Ovaries were scanned by transrectal ultrasonography on d 0, 2, 7, 9, and 11 to assess follicle diameters and ovulation. Blood was collected on d 0, 2, 6, 7, 8, and 9 to quantify serum concentrations of progesterone. Insemination occurred after detected estrus or by timed artificial insemination (TAI) at 64 h after insert removal. Only 25% of 141 GnRH-treated heifers ovulated by d 2; twice as many ovulated when treatment was initiated on d 5 (46.4%) than on other cycle days (20.3%). Diameters of the largest follicle exposed to GnRH on d 0, 2, 7, or 9 did not differ regardless of whether ovulation occurred. Small treatment and stage of cycle differences in diameter of the largest follicle were detected on d 2, 7, and 9. Compared with controls, progesterone concentration was greater in all progesterone-treated heifers on d 2 and 6. Early- vs. late-PGF treatment resulted in less progesterone on d 7 and 8. Pregnancies per AI were less after TAI (44%) than after detected estrus (56%) and were less in controls than in all progesterone treatments. Heifers in which treatments were initiated on d 10 of the cycle had the most consistent (estrus vs. TAI) pregnancies per AI (65.4%) compared with other cycle days. Compared with controls, more progesterone-treated heifers ovulated by 96 h after insert removal. Application of the progesterone insert reduced variance of the interval to estrus after insert removal (or PGF_2α injection in controls) by 1.6-fold compared with controls. These results do not support the use of GnRH in a progesterone-based synchronization protocol.     

Alternative programs to presynchronize estrous cycles in dairy cattle before a timed artificial insemination program

The objective was to test potential presynchronization programs applied to cows before a timed artificial insemination (TAI) program to increase the percentage of cows ovulating in response to both GnRH injections of a TAI program and having a functional corpus luteum before the first GnRH injection of the TAI program. At calving, cows were blocked by lactation (1 vs. 2+) and assigned randomly to receive 1 of 5 presynchronization treatments. Two variants of the standard Presynch program were tested in which 2 injections of PGF_2α were administered 14 d apart with either 14 d (Pre14; n = 122), 12 d (Pre12; n = 123), or 10 d (Pre10; n = 151) intervening before a TAI program was initiated. Two other presynchronization programs consisted of administering a progesterone-releasing controlled internal drug release (CIDR) insert for 7 d plus PGF_2α administration at insert removal. Insert removal occurred either 10 d (CIDR10; n = 157) or 3 d (CIDR3; n = 117) before a TAI program was initiated. The TAI program was a standard Cosynch program [injection of GnRH 7 d before (GnRH-1) and 72 h after (GnRH-2) PGF_2α with TAI administered 72 h after PGF_2α). Cosynch served as the control (n = 157), and cows were assumed to be starting this program at random stages of the estrous cycle. From a subset of cows per treatment (ranging from 49 to 51 cows each), blood samples were collected from coccygeal vessels by using evacuated tubes at d −28, −14, 0 (onset of TAI program), 7, 9, 14, and 21. Ovarian scans were conducted on d 0, 7, 9, 14, and 21 by transrectal ultrasonography. Diameters of follicles and corpus luteum were measured at each exam, and ovulation was determined on d 7 (response to GnRH-1 on d 0) and d 14 (response to GnRH-2 on d 10). Ovulatory incidence after GnRH-1 (47.1 to 67.3%) and GnRH-2 (78 to 90.2%) varied but did not differ among treatments. Before GnRH-1, progesterone concentrations were less in the CIDR3 treatment than in all other treatments. Before GnRH-2, progesterone was greater in the CIDR3 treatment than in all other treatments. Luteal regression and synchronization rate (successful luteolysis and ovulation after GnRH-2) did not differ among treatments. Pregnancy rate per AI at 32 and 60 d post TAI was less in CIDR3 cows than in cows in all other treatments. None of the Presynch treatments improved key responses (ovulation, luteolysis, and synchronization rate) known to improve fertility compared with a standard Cosynch program without presynchronization.     

Effect of dose and timing of prostaglandin F2α treatments during a Resynch protocol on luteal regression and fertility to timed artificial insemination in lactating Holstein cows

Our objective was to evaluate the effect of a second PGF_2α treatment (25 mg of dinoprost) or a double dose of PGF_2α (50 mg of dinoprost) during a Resynch protocol on luteal regression and pregnancies per artificial insemination (P/AI) in lactating dairy cows. Lactating Holstein cows (n = 1,100) were randomly assigned at a nonpregnancy diagnosis to receive (1) Ovsynch (control: 100 µg of GnRH; 7 d, 25 mg of PGF_2α; 56 h, 100 µg of GnRH), (2) Ovsynch with a second PGF_2α treatment (GPPG: 100 µg of GnRH; 7 d, 25 mg of PGF_2α; 24 h, 25 mg of PGF_2α; 32 h, 100 µg of GnRH), or (3) Ovsynch with a double dose of PGF_2α (GDDP: 100 µg of GnRH; 7 d, 50 mg of PGF_2α; 56 h, 100 µg of GnRH). All cows received timed artificial insemination (TAI) approximately 16 h after the second GnRH treatment (G2). Pregnancy diagnosis was performed by transrectal palpation 39 ± 3 d after TAI, and pregnancy status was reconfirmed 66 d after TAI. Blood samples collected from a subset of cows in each treatment at the first PGF_2α treatment (n = 394) and at G2 (n = 367) were assayed for progesterone (P4). Data were analyzed by logistic regression using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC). At 39 d after TAI, GPPG cows tended to have more P/AI than control cows [35% (137/387) vs. 31% (107/349)], whereas P/AI for GDDP cows [32% (118/364)] did not differ from that for control cows. Pregnancy loss from 38 to 66 d did not differ among treatments and was 8% (30/362). The percentage of cows with complete luteal regression (P4 <0.4 ng/mL at G2) tended to differ among treatments and was greater for GPPG cows than for GDDP and control cows (94% vs. 88% vs. 88%, respectively). Overall, cows with P4 <1 ng/mL at the first PGF_2α treatment had fewer P/AI than cows with P4 ≥1 ng/mL (27% vs. 38%), whereas cows with P4 ≥0.4 ng/mL at G2 had fewer P/AI than cows with P4 <0.4 ng/mL (15% vs. 38%). We conclude that adding a second PGF_2α treatment 24 h after the first within a Resynch protocol tended to increase the proportion of cows undergoing complete luteal regression and P/AI, whereas treatment with a double dose of PGF_2α at a single time did not.     

Short communication: Optimization of a timed artificial insemination program for reproductive management of heifers in Canadian dairy herds

Pregnancy per AI (P/AI) following the use of 1 of 2 timed AI (TAI) protocols and 2 different intervals between TAI and resynchronization were compared in heifers that were inseminated with either conventional or sex-sorted semen. Holstein heifers (n = 317; 527 inseminations) were submitted to a 5-d Cosynch protocol with (+) or without (–) GnRH at the time of controlled internal drug release (CIDR) insertion on d 0, CIDR removal and a single PGF_2α treatment on d 5, and TAI plus GnRH on d 8 (72 h later). Visual estrus detection (ED) was conducted on d 6 in the afternoon and d 7 in the morning and heifers observed in estrus were artificially inseminated on d 7 in the afternoon. Heifers were alternately assigned conventional or sex-sorted semen. Pregnancy was diagnosed by ultrasound 27 and 42 d after AI, and heifers diagnosed as nonpregnant were resynchronized, up to 3 times, starting on d 27 or 34 to provide an interbreeding interval of 35 or 42 d. Overall, TAI protocol had no effect on P/AI at 27 or 42 d after artificial insemination or on pregnancy loss, but P/AI following the first service tended to be higher in the –GnRH TAI group (66.3 vs. 56.8%). Pregnancy per AI at 27 d (61.9 vs. 55.5%) tended to differ between conventional and sex-sorted semen. Heifers artificially inseminated based on ED tended to have a greater P/AI (67.6 vs. 58.2%) and had decreased pregnancy loss (0.0 vs. 4.1%) than those submitted to TAI. A greater number of heifers in the –GnRH TAI protocol were artificially inseminated on ED than the +GnRH TAI protocol (21.5 vs. 13.7%). No difference in P/AI was observed between the 35- and 42-d interbreeding intervals; however, more heifers in the 42-d group were artificially inseminated based on ED than in the 35-d group (22.7 vs. 7.8%). A 5-d Cosynch+CIDR TAI protocol without the initial GnRH and with a single PGF_2α at CIDR removal is an acceptable alternative to achieve high P/AI when either conventional or sex-sorted semen is used in Holstein heifers. Breeding heifers based on detected estrus increases labor, but has the potential to increase fertility.     

Effects of additional prostaglandin F2α and estradiol-17β during Ovsynch in lactating dairy cows

This study was designed to evaluate whether decreasing circulating progesterone (P4) or increasing circulating estradiol-17β (E2) near the time of artificial insemination (AI) in an Ovsynch protocol would increase pregnancies per AI (P/AI) in lactating dairy cows. Six hundred nineteen lactating Holstein cows (n = 772 inseminations) received Ovsynch (GnRH-7 d-PGF_2α-56 h-GnRH-16 h-timed AI). Cows were randomized in a 2 × 2 factorial experiment of 4 treatments to receive or not receive 25 mg of PGF_2α 24 h after the standard PGF_2α of Ovsynch, or 0.5 mg of E2 at the time of the final GnRH of Ovsynch, or both. Blood samples were collected 24 h after normal PGF_2α and at final GnRH to evaluate circulating P4. Ovarian ultrasound was done at final GnRH to determine preovulatory follicle size. Ovulation was confirmed by ultrasound 5 d after AI. Treatment with additional PGF_2α increased the percentage of cows that had complete luteal regression (95.6%) compared with control cows (84.6%). In contrast, additional PGF_2α had no detectable effect on P/AI (control = 41.5% vs. + PGF_2α = 44.7%). Supplementation with E2 increased expression of estrus (84.4 vs. 37.2%), but had no effect on overall fertility and even tended to have a negative effect on fertility in cows that ovulated to the second GnRH (control = 51.5% vs. +E2 = 44.0%). Thus, additional treatments with PGF_2α or E2 during Ovsynch can be used to increase synchronization and expression of estrus during Ovsynch, although the lack of improvement in fertility makes these treatments unwarranted.     

Detection of anovulation by heatmount detectors and transrectal ultrasonography before treatment with progesterone in a timed insemination protocol

Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol [with or without progesterone supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF_2α 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF_2α injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone ≥1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.     

Factors influencing upfront single-and multiple-ovulation incidence, progesterone, and luteolysis before a timed insemination resynchronization protocol

Our objectives were to determine relationships among factors influencing responses to the first GnRH injection in a timed artificial insemination (TAI) protocol and subsequent fertility after altering timing of the second GnRH injection and AI relative to PGF_2α injection. Replacement heifers (n = 86) and 613 lactating cows previously inseminated were diagnosed not pregnant to form 77 breeding clusters spanning 36 mo. At not-pregnant diagnosis (d 0), females received 100 μg of GnRH, and then 7 d later, they received 25 mg of PGF_2α. Females in 2 treatments received GnRH 48 h (G48) after PGF_2α injection and TAI at the time of the second GnRH injection (G48 + TAI48) or 24 h later (G48 + TAI72). Females in the third treatment received GnRH 72 h after PGF_2α when inseminated (G72 + TAI72). Neither timing of GnRH nor time of AI altered TAI pregnancy rates (average of 20.4%). Ovaries of females in 65 clusters were scanned on d 0 (first GnRH injection) and 7 d later (PGF_2α injection). Ovarian structures were mapped and ovulation in response to the first GnRH injection was evaluated on d 7. When estrus was detected before scheduled TAI, females were inseminated; otherwise, TAI conception of remaining females was based on timing of GnRH and AI in 3 treatments. On d 7, 1 or more new corpora lutea (CL) were detected in 43% of females and their pregnancy rate was subsequently greater (28 vs. 18%) than those not ovulating. Follicle diameters on d 0 did not differ between females that did (11.9 ± 0.3 mm) and did not (11.8 ± 0.4 mm) subsequently ovulate in response to GnRH. Follicle diameter and number of follicles ≥5 mm increased with increasing lactation number, but decreased with increasing number of CL. Diameter of follicles in which more than 1 follicle ovulated decreased linearly from that in which only 1 follicle ovulated. Incidence of ovulation increased with increasing lactation number and total number of follicles ≥5 mm, but decreased with increasing number of CL. Incidence of multiple ovulations (15%) was greater in females having more follicles ≥5 mm and in those in early diestrus. Multiple ovulation did not occur in heifers, but was decreased in cows having more than 1 CL. In cows having more than 1 CL, luteal regression was reduced by 5.6 percentage units compared with those having 1 CL. In a TAI protocol, pregnancy rate was greater for females in early diestrus compared with females in other stages of the cycle, in those that ovulated after the first GnRH injection, in those having luteolysis, and in those inseminated during nonsummer months.     

Spatial relationships of ovarian follicles and luteal structures in dairy cows subjected to ovulation synchronization: Progesterone and risks for luteolysis,ovulation, and pregnancy

Objectives were to determine relative ovary location of follicles, GnRH-induced corpora lutea (CL), and older CL present in ovaries as part of ovulation synchronization and their associations with progesterone concentration and risk for luteolysis, ovulation, and pregnancy. Cows were exposed to a timed artificial insemination (AI) program [GnRH-1–7 d–PGF_2α (1 dose or 2 doses 24 h apart)–56 h after first or only dose of PGF_2α–GnRH-2–16 h–timed AI at 72 ± 3 d in milk]. Blood was collected to assess progesterone when ovarian structures were mapped in 694 cows before GnRH-1 and before and 48 h after PGF_2α and, in a subset of cows, size of CL (n = 599) and progesterone (n = 380) at 6 d after AI. Dominant follicles and CL in single-ovulating cows were detected more often in right than left ovaries (follicles before GnRH-1: 60.6% right and GnRH-2: 61.2% right; and CL before GnRH-1: 58.6% right and GnRH-2: 66.4% right). Dominant follicles in single-ovulating cows before GnRH-1 tended to be ipsilateral to the CL more often than contralateral (54.8 vs. 45.2%) with co-dominant follicles identified in both ovaries (19.3%). In response to GnRH-1 or GnRH-2, more left-ovary follicles ovulated contralateral to CL (left to right, 54.7%; right to left, 34.7%) than right-ovary follicles, but fewer left-ovary follicles ovulated ipsilateral to CL (left to left: 45.3%) than right-ovary follicles ovulated ipsilateral (right to right: 65.3%). Preovulatory follicles in single-ovulating cows before PGF_2α tended to be detected more often ipsilateral than contralateral to CL induced by GnRH-1 (younger CL; 56.5 vs. 43.6%), but were of equal frequency ipsilateral or contralateral to older CL present before GnRH-1. Luteolytic risk was less in cows bearing co-dominant follicles in both ovaries compared with those in either right or left ovaries. Luteolytic risk in single-ovulating cows did not differ between ovaries. Luteolytic risk was greater for cows bearing older CL (86.5%) than for cows bearing younger GnRH-1-induced CL (65.3%) or both (79.6%). Pregnancy risk at 60 d after AI was or tended to be greater in cows having both CL types compared with either younger or older CL, respectively, partly because of greater embryonic loss in the latter 2 cases. More female calves tended to be carried in right horns when conception occurred after first service, whereas the opposite greater female frequency occurred in left horns after repeat services. Right-ovary dominance is evident before and after GnRH treatment.     

Pregnancy per artificial insemination in lactating dairy cows subjected to 2 different intervals from presynchronization to initiation of Ovsynch protocol

A protocol for presynchronization of ovarian status with 2 injections of PGF_2α given 14 d apart, with the last PGF_2α injection given 12 or 14 d before Ovsynch increases pregnancy per artificial insemination (P/AI) in dairy cows. We determined the efficacy of reducing the interval from the last PGF_2α injection (500 μg of cloprostenol) of presynchronization to initiation of Ovsynch on response to treatment and P/AI. Lactating dairy cows were assigned to an Ovsynch protocol, with the initial injection of GnRH given either 9 (PRE-9; n = 135) or 12 d (PRE-12; n = 135) after the second PGF_2α injection of presynchronization. The Ovsynch protocol consisted of 2 injections of 100 μg of GnRH given 9 d apart and 1 injection of PGF_2α given 7 d after the initial GnRH injection, and cows were subjected to timed artificial insemination (TAI; 70 ± 3.5 DIM) approximately 16 h after the second GnRH injection. Body condition score (1–5 scale) was recorded at TAI. Blood samples were taken for progesterone determination at the PGF_2α injection of Ovsynch, at TAI, and at 11 d after TAI. Ultrasonographic examinations were done in all cows at the second PGF_2α injection of presynchronization, initial GnRH injection, PGF_2α injection of Ovsynch, at TAI, and 24 h after TAI for cyclicity status and ovarian responses to treatments, and at 32 and 60 d after TAI for confirmation of pregnancy. Overall, 29 cows (10.7%) were determined acyclic or cystic and excluded from the study. The percentage of cows responding to initial GnRH injection (62.2 vs. 61.5%) did not differ between PRE-9 and PRE-12 but more cows in the PRE-9 group failed to respond to PGF_2α treatment of Ovsynch compared with PRE-12 (22.7 vs. 10.7%). Body condition score at TAI (2.9 ± 0.02) and mean ovulatory follicle diameter (16.4 ± 0.2 mm) were not different between treatments. Overall P/AI at 32 d was reduced in PRE-9 (33.6%) compared with PRE-12 (44.3%) but pregnancy losses (5.0 vs. 3.7%) did not differ between treatments. Primiparous cows in the PRE-12 group had higher mean progesterone concentration 11 d after TAI and greater P/AI 32 after TAI than primiparous cows in the PRE-9 group (6.4 ± 0.5 vs. 4.6 ± 0.5 ng/mL and 55.8 vs. 30.0%, respectively). In conclusion, reducing the interval from the last PGF_2α injection of the presynchronization treatment to initiation of Ovsynch (from 12 to 9 d) did not affect ovulatory response to initial GnRH injection but reduced response to PGF_2α injection of Ovsynch and P/AI at 32 and 60 d after TAI. The reduction in P/AI was particularly evident in primiparous cows of the PRE-9 group.     

Presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously improved first-service pregnancy per artificial insemination in lactating Holstein cows compared with Presynch-14 when combined with detection of estrus

This study aimed to determine the effect of 2 simple breeding strategies combining artificial insemination (AI) after detection of estrus (AIED) and timed AI (TAI) on first-service fertility in lactating Holstein cows. Weekly, lactating Holstein cows (n = l,049) between 40 and 46 d in milk (DIM) were randomly assigned to initiate 1 of 2 breeding strategies for first service: Presynch-14 and PG+G. Presynch-14 is a presynchronization strategy with 2 PGF_2α treatments 14 d apart with the last PGF_2α 14 d before the initiation of the Ovsynch protocol. Cows treated with PG+G receive a simpler presynchronization program that uses PGF_2α and GnRH simultaneously 7 d before Ovsynch. In both treatments, cows detected in standing estrus by tail chalk at any time ≥55 DIM were inseminated, and treatment was discontinued (n = 525). Cows completing treatment received TAI from 78 to 84 DIM (n = 526). In a subgroup of cows that received TAI, blood was collected (n = 163) to assess circulating concentrations of progesterone, and ultrasonographic evaluations of ovaries were performed on the day of first GnRH of Ovsynch (n = 162) and PGF_2α of Ovsynch (n = 122). The proportion of cows that received TAI was greater for PG+G compared with Presynch-14 (63.5 vs. 31.9%), which increased DIM at first service for cows treated with PG+G compared with Presynch-14 (75.5 ± 0.4 vs. 68.7 ± 0.4). For cows receiving TAI, the ovulatory response to first GnRH of Ovsynch (73.8 vs. 48.8%) and the proportion of cows with functional corpora lutea (92.6 vs. 73.1%) were greater for PG+G than Presynch-14. Cows treated with PG+G had greater overall pregnancy per AI (P/AI) 42 ± 7 d after AI (40.2 vs. 33.6%) and calving per AI (32.1 vs. 25.2%) than Presynch-14. For cows receiving AIED, treatment did not affect P/AI 42 ± 7 d after AI. However, for cows receiving TAI, PG+G increased P/AI compared with Presynch-14 (44.6 vs. 35.2%). Overall, cows receiving TAI had greater P/AI 42 ± 7 d after AI (42.5 vs. 31.5%) and calving per AI (34.1 vs. 23.7%) and decreased pregnancy loss (16.8 vs. 25.2%) than cows receiving AIED. In summary, PG+G increased the proportion of cows receiving TAI and the DIM at first service, P/AI, and calving per AI compared with Presynch-14 when both TAI programs were combined with AIED.     

Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows

Objectives were to develop a timed artificial insemination (TAI) resynchronization program to improve pregnancy per AI and to evaluate responses of circulating progesterone and pregnancy-associated glycoproteins in lactating cows. Cows (n = 1,578) were presynchronized with 2 injections of PGF_2α, given 14 d apart starting on d 45 ± 3 postpartum, followed by Ovsynch [2 injections of GnRH 7 d before and 56 h after injection of PGF_2α, TAI 16 h after second injection (d 0)]. The Resynch-treated cows received an intravaginal progesterone insert from d 18 to 25, GnRH on d 25, and pregnancy diagnosis on d 32, and nonpregnant cows received PGF_2α., GnRH 56 h later, and TAI 16 h later (d 35). The control cows were diagnosed for pregnancy on d 32 and nonpregnant cows received GnRH, PGF_2α 39 d after TAI, GnRH 56 h later, and TAI 16 h later (d 42). Pregnancy was reconfirmed on d 60 after AI. Ovarian structures were examined in a subset of cows at the time of GnRH and PGF_2α injections. Blood samples for analyses of progesterone and pregnancy-associated glycoproteins were collected every 2 d from d 18 to 30 in 100 cows, and collection continued weekly to d 60 for pregnant cows (n = 43). Preenrollment pregnancies per AI on d 32 did not differ for cows subsequently treated as Resynch (45.8%, n = 814) and control (45.9%, n = 764), and pregnancy losses on d 60 were 6.7 and 4.0%, respectively. Resynchronized service pregnancy per AI (36%, n = 441; 39.5%, n = 412) and pregnancy losses (6.3 and 6.7%) did not differ for Resynch and control treatments, respectively. Days open for pregnant cows after 2 TAI were less for the Resynch treatment than for the control treatment (96.2 ± 0.82 vs. 99.5 ± 0.83 d). Cows in the Resynch treatment had more large follicles at the time of GnRH. The number of corpora lutea did not differ between treatments at the time of PGF_2α. Plasma progesterone for pregnant cows was greater for Resynch cows than for control cows (18-60 d; 6.6 vs. 5.3 ng/mL), and plasma concentrations of progesterone on d 18 were greater for pregnant cows than for nonpregnant cows (5.3 vs. 4.3 ng/mL). Plasma pregnancy-associated glycoproteins during pregnancy were lower for cows in the Resynch treatment compared with control cows on d 39 (2.8 vs. 4.1 ng/mL) and 46 (1.3 vs. 3.0 ng/mL). Cows pregnant on d 32 that lost pregnancy by d 60 (n = 7) had lower plasma concentrations of pregnancy-associated glycoproteins on d 30 than cows that maintained pregnancy (n = 36; 2.9 vs. 5.0 ng/mL). Pregnancy-associated glycoproteins on d 30 (>0.33 ng/mL) were predictive of a positive d 32 pregnancy diagnosis (sensitivity = 100%; specificity = 90.6%). In conclusion, Resynch and control protocols had comparable pregnancy per AI for first and second TAI services, but pregnancy occurred 3.2 d earlier in the Resynch group because inseminations in the Resynch treatment began 7 d before those in the control treatment. Administration of an intravaginal progesterone insert, or GnRH, or both increased progesterone during pregnancy. Dynamics of pregnancy-associated glycoproteins were indicative of pregnancy status and pregnancy loss.